Recombinant antigens of the porcine circovirus 2 (pcv-2) for vaccine formulations and use thereof

ABSTRACT

The present continuation-in-part of the Application BR 7 10 2013 001893 refers to the obtainment of the viral capsid recombinant antigen of the Porcine circovirus 2 (PCV-2) and modifications thereof, upon expression in prokaryotic system, recovery of virus-like particles (VLPs) and its use in vaccine formulations. The antigens and vaccine formulations can be used in the immunization of animals in control programs of the diseases associated with PCV-2 in conventional swine production systems and represent alternatives to vaccines available on the market.

The innovations and improvements herein refer to the removal of theviral capsid protein coding sequence of the porcine circovirus 2 (PCV-2)and transferred to a bacterial expression vector. This plasmid constructallows the expression of the recombinant protein without the10-histidine tail. The expression of virus-like particles (VLP) by therecombinant protein was also verified. Assays were performed in murineand swine models, where the immunogenicity of the vaccine candidate ofthe patent object was confirmed.

DESCRIPTION OF THE DRAWINGS

The FIG. 1 shows the analysis in agarose gel 1% of the cleavagereactions of the empty pET29a (channel 1), the cleavage of therecombinant plasmid pCap-rPCV2-29a with the XhoI and NdeI enzymes(channel 2), M: Ladder DNA marker 100 bp.

The FIG. 2 confirms the expression of rCAP-PCV-2 from thepCAP-rPCV2-29a. SDS-PAGE Gel 15% (left) and nitrocellulose membraneobtained by Western blotting (right). M: molecular weight marker; C−:Negative control (soluble fraction of the E. coli extract transformedwith the empty bacterial expression plasmid and induced with IPTG); and3: Sample (soluble fraction of the E. coli extract transformed with thepCAP-rPCV2-29a and induced with IPTG). The arrow indicates the band ofapproximately 27 kDa corresponding to rCap-PCV-2.

The FIG. 3 shows the fractions resulting from the precipitation processof the soluble fraction by saturation with ammonium sulfate. M:Molecular weight marker; 1: Soluble fraction—part not precipitated; 2:purified rCAP. The arrow indicates the RCAP-PCV2-29a.

The FIG. 4 show electron micrographs: formation of virus-like particles(VLPs) by rCap-PCV2-29a without histidine tail. The 200 mesh grids withformvar/carbon containing samples were analyzed by Transmission ElectronMicroscope using 85000× (left figure) and 140000× (middle and rightdrawings) magnifications. VLPs can be visualized by the arrows.

The FIG. 5 shows the specific humoral response induced by the rCap-PCV-2in Balb/c mice before and after vaccination as measured by indirectELISA. The animals were inoculated on days 0 and 21. The inoculatedcandidate containing the rCap-PCV-2 lacking histidine tail (G4) showedhigh levels of antibodies compared to controls (G1 and G2) used duringthe collections 2 (28 days) and 3 (42 days) of the experiment.

The FIG. 6 shows IFN-γ dosage by ELISA obtained through thelymphoproliferation assay of spleen cells from the various experimentalgroups stimulated with concanavalin A (ConA) as positive control, RPMImedium supplemented with bovine fetal serum as negative control andpurified rCap-PCV-2 in concentrations of 0.05 μg/mL, 0.5 μg/mL and 5.0μg/mL in the period of 72 hours. It was observed that the groupinoculated with the proposed vaccine formulated (G4) when stimulatedwith 5.0 μg/mL of the purified PCV-2-rcap presented statisticaldifference compared to controls. It is noteworthy that the vaccinecandidate induced the IFN-γ production, which is a cytokine of theT_(H)1 response profile and promoted a more efficient cellular immuneresponse against the virus.

The FIG. 7 shows IL-10 dosage by ELISA obtained by thelymphoproliferation assay of splenic cell of the different experimentalgroups stimulated with concanavalin A (ConA) as positive control, RPMImedium supplemented with bovine fetal serum as negative control andpurified rCap-PCV-2 in concentrations of 0.05 μg/mL, 0.5 μg/mL and 5.0μg/mL in a period of 72 hours. It was observed that the group inoculatedwith the proposed vaccine formulated (G4) showed statisticallysignificant IL-10 levels compared to controls, for both positive stimuli(ConA) and different concentrations of rCap-PCV-2. IL-10 is acharacteristic cytokine of the T_(H)2 response profile(immunosuppressive) and inhibits the synthesis of pro-inflammatorycytokines such as IL-12, thus directing the response to humoralimmunity.

The FIG. 8 shows IL-12 dosage by ELISA obtained through thelymphoproliferation assay of spleen cells from the various experimentalgroups stimulated with concanavalin A (ConA) as positive control, RPMImedium supplemented with bovine fetal serum as negative control andpurified rCap-PCV-2 in concentrations of 0.05 μg/mL, 0.5 μg/mL and 5.0μg/mL in a period of 72 hours. Low quantification was observed for theIL-12 cytokine and there was no significant difference in the groupinoculated with the vaccine candidate (G4) to the control groups. Thelow quantification of IL-12, a characteristic cytokine of the T_(H)1response profile, is due to increased IL-10 levels (FIG. 7) which is animmunosuppressive cytokine, and tends to inhibit the production ofinflammatory cytokines, which are characteristics of T_(H)1 cells. Itwas observed that when quantifying the levels of different cytokines,the immune response mediated by the immunization with the vaccinecandidate shown to be of the mixed type, i.e. there was a nopredominance of T_(H)1 or T_(H)2 response.

The FIG. 9 shows the serological profile detected during the trialperiod in the farm I during the different collections. On the horizontalaxis, the four collections are presented: Collection 1—Blood samplescollected at 21 days old (day 0 post-vaccination); Collection 2—Bloodsamples collected at 53 days old (32 days post-vaccination); Collection3—Blood samples collected at 102 days old (81 days post-vaccination);Collection 4—Blood samples collected at 166 days old (145 dayspost-vaccination). On the vertical axis are the levels of antibodiesexpressed in S/P. S/P=(test sample average−negative controlaverage)/(positive control average−negative control average). The testgroup (G1) corresponds to the group that was inoculated with theformulation based on plasmid 29a, at a concentration of 100 μg. Thepositive control group (G2) corresponds to animals inoculated with thecommercial vaccine. The negative control group (G3) are the animals thatwere inoculated with PBS. (*) Indicates statistical difference comparedto the negative control (C⁻). Therefore, the test formulations, G1,resulted in significantly higher levels than the positive and negativecontrols (G2 and G3), on the third and fourth sampling.

The FIG. 10 shows the serological profile observed during the trialperiod in the farm II during the different collections. The horizontalaxis shows the four collections: Collection 1—Blood samples collected at23 days old (day 0 post-vaccination); Collection 2—Blood samplescollected at 65 days old (42 days post-vaccination); Collection 3—Bloodsamples collected at 116 days old (93 days post-vaccination); Collection4—Blood samples collected at 158 days old (135 days post-vaccination).The vertical axis shows the levels of antibody expressed as S/P.S/P=(test sample average−negative control average)/(positive controlaverage−negative control average). The test group G1 corresponds to thenegative control group. The animals in this group were vaccinated withPBS. The test group G2 corresponds to the positive control group. Theanimals in this group were immunized with commercial vaccine. The testgroup G3 corresponds to the group of animals that received the researchvaccine on the soluble fraction derived formulation. In addition, the G4test group corresponds to the group of animals that received theresearch vaccine purified with ammonium sulfate. (*) Indicatesstatistical difference compared to the negative control (C⁻). Therefore,the test formulations, soluble fraction, and ammonium sulfate purifiedvaccine generated significantly higher levels than the negative controlsince the second collection, keeping this behavior until the fourth andfinal collection.

DETAILED DESCRIPTION OF THE INVENTION Transferring of the Capsid ProteinCoding Region of the Porcine Circovirus 2 to the Expression Vector inBacterial System

After verification of the correct sequence of the insert in the vector,the sample of the pCapPCV-2 plasmid DNA, SEQ ID NO: 01 (amplificationvector) was subjected to an enzymatic assay where specific restrictionsites were used to insert the gene ORF2 in another bacterial expressionvector (pET-29a—Novagen). This expression vector is controlled by a T7lac promoter, however the insert was directed so that the recombinantprotein to be coded did not have the sequence encoding the 10-histidinetail in the N-terminal region, as in the vector earlier mentioned in BR10 2013 001893 7, thereby targeting an increased production ofvirus-like particles (VLP's). After the cleavages, the products of therecurring digestions were liked using the enzyme T4 DNA ligase. Theproduct of this liking reaction was then used to transform E. coli DH5α.Thus, the transformants clones were randomly selected from the coloniesfor the identification of the plasmids with the insert, and, to confirmthe cloning, colonies PCRs were performed. The positive colonies wereselected and each colony was subjected to PCR separately. Thisidentification, in turn, is given by digestion reaction with therestriction enzymes XhoI and NdeI restriction. The same digestionreaction was also carried out with the pET29a without the ORF2 gene(empty). All the digestion reactions assays were carried out byelectrophoresis in 1% agarose gel. Thus, the bands of the expected sizewere observed in 718 bp and 5371 bp, from the digestion with NdeI andXhoI, respectively, and a high molecular weight fragment correspondingto the plasmid remaining (FIG. 1). The recombinant plasmids obtainedwere named PCAP-rPCV2-29^(a), and the nucleotide and amino acid sequenceconfirmed as SEQ ID NO: 01 and SEQ ID NO: 02, respectively. Plasmidswere stored in microcentrifuge tubes containing glycerol from 15 to 30%and kept at −80° C.

Expression of the Capsid Recombinant Protein of the Porcine Circovirus 2and Analysis on SDS-PAGE Gel

The total expression of the recombinant proteins was done in mediumscale in 1000 mL of TB (tryptone 12 g/L, yeast extract 24 g/L, glycerol4 mL, monobasic potassium phosphate 2.31 g/L and dibasic potassiumphosphate 12.54 g/U). For this, competent bacteria of the strain E. coliBL21-DE3-RIL codon plus were transformed with the pCap-rPCV2-29aconstruct analogous to that carried out with the amplification vector.Thus, approximately 20 nanograms of recombinant plasmid pCap-rPCV2-29awere added to 100 μL of competent cells and the mixture incubated on icefor 30 min. Then, the cells mixture and plasmid DNA were subjected to athermal shock in a water bath at 42° C. for 1 minute, and again on icefor 2 minutes. Thereafter, 900 uL of LB medium (bacto-tryptone 10 g/L,yeast extract 5 g/L and sodium chloride 10 g/L) without antibiotic wasadded and cells incubated at 37° C. for 2 hours at 250 rpm. The cellswere diluted hundred-fold (1:100) into LB medium containing kanamycin 50μg/mL and incubated at 37° C. and 180 rpm for 12-16 hours(pre-inoculation). A negative control culture (same bacteria of strainE. coli BL21-DE3-RIL codon plus but not transformed) was also performedin liquid LB, pH 7.0, chloramphenicol 17 mg/mL. The cells were thendiluted 1:100 in TB liquid medium, pH 7.0, kanamycin 50 μg/mL and theculture was grown at 30° C./180 rpm for approximately 4 hours until theoptical density (OD₆₀₀) of from 0.6 to 0.8. It was performed the sameway as for the negative control, using chloramphenicol 17 mg/mL. Afterreached the OD₆₀₀, IPTG was added to final concentration of 0.25 mM forthe expression of the recombinant protein of interest, the cultures werethen left at a temperature of 30° C. for additional 4 hours, alwaysunder vigorous agitation and sufficient aeration. The same procedure wascarried out for the negative control. After induction in optimalconditions, the samples from the inductions were centrifuged at 10,000 gfor 20 min at 4° C. The supernatants were discarded and the precipitatedcells were stored at −20° C.

The precipitate resulting from a volume of 100 mL of the induced mediumof the pCap-rPCV2-29a was thawed and resuspended in lysis buffer(NaHCO₃50 mM, NaCl 60 mM, pH 7.3) to a final volume of approximately 5mL. The process of cell lysis was performed with 6 cycles of 10 ssonication at 200-300 watts each, with intervals of 10 s and with thetubes on ice to prevent the sample warming. The cellular debris and theinclusion bodies were precipitated by centrifugation at 15,000×g for 30min at 4° C. The supernatant (soluble fraction) was collected in a newtube and used for the purification of capsid recombinant protein ofPCV-2, referred to herein as rCap-PCV2 (SEQ ID NO: 02) derived from thepCap-PCV2-29a induction.

The samples (including negative controls) were analyzed inpolyacrylamide gel 15% (Sambrook J., Russell D. W., Molecular Cloning: Alaboratory manual, 3^(rd) ed., Cold Spring Harbor Laboratory Press, NewYork, 2001). After the running, the gel was revealed by stainingsolution (Coomassie Brilliant Blue R-250 0.1%, acetic acid 9%, ethanol45%). The electrophoresis analysis confirmed the presence of a band ofapproximately 27 kDa corresponding to the mass of the protein encoded bythe ORF2 without the histidine tail (rCap-PCV2-29a). The confirmation ofthe expression was given by Western blotting technique (FIG. 2).

Purification of the Capsid Recombinant Protein of the Porcine Circovirus2 Expressed in pET29a Plasmid and Analysis on SDS-PAGE Gel

For the recombinant protein (rCap-PCV2-29a) from the pCap-rPCV2-29a, thepurification of the soluble fraction was performed by precipitation withammonium sulphate 20%-40%. The soluble fraction was left under 180 rpmstirring at 0° C. for 2 hours. After this step, the sample wascentrifuged at 10000×g at 4° C. for 30 minutes. Subsequently, thesupernatant was separated and the precipitate resuspended in carbonatebuffer (NaCl 60 mM; NaCO₃H 50 mM; pH 7.3). The analysis of the resultswas made by polyacrylamide gel 15% (FIG. 3). The amount of recombinantprotein in the purified PCV-2 viral capsid was determined using themethod described by Bradford (Bradford, M. M., 1976, A rapid andsensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dyebinding. Analytical biochemistry,V. 72, p. 248-254, 1976). The calculation was made by linear regressionwhere the equation y=0.3267+0.0108x was obtained from the best fit tothe optical density values for the tested BSA dilutions.

Preparation of the Vaccine Compound and Verification of the Virus-LikeParticles (VLPs) Formation

The purification of pCap-PCV2-29a in the soluble fraction from thepCap-rPCV2-29a was performed by precipitation with ammonium sulfate at20% to 40%. The precipitate was resuspended in carbonate buffer (NaCl 60mM; NaCO₃H 50 mM; pH 7.3) (FIG. 3). This sample was then used to preparethe vaccine candidate, where this preparation is given by the additionof an adjuvant in a previously quantized amount of sample.

For VLPs verification, fractions from the CsCl gradient that showed apositive result on the Western-blotting were dialyzed separately against500 mL of carbonate buffer (NaCl 300 mM, bicarbonate 50 mM, pH 7.0) twotimes for 4 hours each. Approximately 10 μL of each fraction were addedto the 200 mesh grids covered with formvar/carbon and allowed to settlefor 1 minute at room temperature. Then, the excess sample was removedwith filter paper and a drop of uranyl acetate 2% was added in each gridand allowed to act for 1 minute. The excess of that contrast was removedwith filter paper and the grids were left in a desiccator for 2 days.The analysis was performed in a transmission electron microscope and theimages were photographed with 85000× and 140000× amplification. Theresults from this analysis can be seen in the three images of FIG. 4. Alarge production of VLPs was observed in the soluble fraction and forthe vaccine formulated containing the pCap-PCV2-29a, which demonstratesthat the histidine tag removal favored a larger production of virus-likeparticles in the samples.

Demonstration Experiments Experiments in Mice

25 female mice Balb/c of approximately 5 weeks old were used from thevivarium connected to the Life Sciences and Health Center (CCB) of theFederal University of Viçosa (UFV), which were equally divided into 5groups. The procedures were performed according to the Animal Ethics andExperimentation Committee of the Federal University of Vigosa (UFV).

The group 1 (G1) was vaccinated with PBS 1× (negative control), thegroup 2 (G2) was vaccinated with the commercial vaccine (positivecontrol), the group 3 (G3) was vaccinated with the rCap protein purifiedin FPLC (rCap-PCV2), the group 4 was vaccinated with the vaccineformulated produced from pCap-rPCV2-29a plasmid. It should be noted thatin this assay the recovery of the vaccines antigen for the vaccinationof animals in the G4 group was performed by precipitation withpolyethylene glycol (PEG6000) due to of its ability to purify viralparticles. To the soluble fractions obtained after bacterial lysisprocess were added PEG₆₀₀₀ solutions in previously standardizedconcentrations for the rCap-PCV-2-29a. The tubes containing the antigento be recovered together with the PEG₆₀₀₀ were cooled and left understirring for the viral particles precipitation. After this step, thesamples were centrifuged and the precipitates were suspended in lysisbuffer.

With the exception of the group inoculated with commercial vaccines, thevaccines were administered with aluminum hydroxide adjuvant at aconcentration of 1 mg/mL. The animals were vaccinated twice (two doses)subcutaneously at an interval of 21 days between doses. The amount ofrCap-PCV2 vaccinated was 50 μg for the first dose and 25 μg for thesecond dose. Blood samples were collected via ocular sinus puncturebefore each inoculation, corresponding to the days 0, 28, and 42 for theserological analysis. The evaluation of the humoral immune response wasmade by indirect ELISA technique. The optimum working concentrations ofthe antigen rCap-PCV-2 and the best dilution of serum (primary antibody)were evaluated by Checker board titrating (Crowther J. R. ELISA. Theoryand Practice. Methods in Molecular Biology. V. 42, p. 1-223, 1995). Itwas determined as the optimal antigen concentration (rCap-PCV-2), 28μg/well and 1:100 as the ideal serum dilution (primary antibody). Theanalyses were performed using a positive sample of mouse serum forPCV-2.

The mice vaccinated with the vaccine candidate containing therCap-PCV2-29a (without histidine tail) had antibody levels higher thanthose obtained for mice vaccinated with commercial vaccines in periodscorresponding to collections 2 (28 days) and 3 (42 days), as shown inFIG. 5. The presence of VLPs in the proposed vaccine candidate (FIG. 4)possibly contributed to a better immune response in this group ofanimals.

The mice from all groups were euthanased and the spleen were removedaseptically and divulsed in EDTA 200 mM to obtain total spleen cells.These cells were washed with RPMI 1640, supplemented with streptomycin1.00 g/L and penicillin 0.75 g/L, centrifuged at 1.000×g for 10 min at4° C. for 4 min and incubated with lysis buffer (9 parts of ammoniumchloride 0.16 M and 1 part of Tris-HCl 0.17 M) at room temperature. Thecells were again washed and suspended to a concentration of 1×10⁶cells/mL in RPMI medium supplemented with fetal bovine serum 5% (FBS).Subsequently, the cells were added to 24-well plates (in duplicate foreach experimental group) (1000 μL/well) and incubated at 37° C. underatmosphere of CO₂ 5% 72 hours using the treatments with RPMI mediumsupplemented with FBS 5% (negative control), with concanavalin Astimulation (ConA 2 μg/mL) used as positive control, and with therecombinant capsid protein of PCV2 purified on affinity column atconcentrations of 0.05 μg/ml, 0.5 μg/mL and 5.0 μg/mL. After theincubation period, the plates were centrifuged at 1000×g for 5 min forcells sedimentation and the supernatants collected for evaluation of thecytokines profile by ELISA.

The supernatants collected in the lymphoproliferation concerned to theperiod of 72 hours were analyzed by ELISA for evaluation of the T_(H)1and T_(H)2 profile. The Murine IFN-gamma ELISA kit, Murine IL-12 ELISAkit, Murine IL-10 ELISA Kit and Murine IL-4 ELISA kit commercial kitswere used for the Tel profile evaluation: IL-12 and IFN-γ and T_(H)2profile: IL-4 and IL-10 (PeproTech Brazil—FUNPEC), according to themanufacturer's manual.

As can be seen in FIGS. 6 to 8 the dosage of the INF-γ, IL-10 and IL-12cytokines in ng/mL showed a mixed response profile, i.e. not beingpredominantly T_(H)1 or T_(H)2. High levels of INF-γ favor a T_(H)1profile response in which the cellular immunity, essential for combatingintracellular pathogens (virus), is preferred. However, the largestquantifications for IL-10 indicate a direction of the immune response toT_(H)2 profile, where the antibody production is favored. This resultconfirms what was observed in FIG. 5, indicating a higher serologicallevel for mice vaccinated with the proposed vaccine candidate(G4—rCap-PCV-2-29a) in relation to the controls (G1 and G2) used in thisexperiment.

Trial in Swine Farm I

The Trial I was conducted in a commercial farm of complete cycle,naturally infected, in the region of Zona da Mata—MG respecting themanagement adopted by the farm. This study was conducted in accordanceto the ethical standards of animal testing and requirements of theEthics Committee on Animal Use (CEUA) of the UFV. 105 swine were used,males and females, Choice Genetics commercial line, approximately 21 dayold, divided into 3 groups of 35 animals.

At 21 days of age, the piglets were identified with an ear tag properfor swine and subsequently weighed for the of group division. Thisdivision was done by random experimental lineation, so that all groupshad similar mean weight. The immunization was performed by intramuscularroute with doses of 1 mL in the piglets with an average age of 21 days,so that the group 1 received the formula from the pCap-PCV2-29a proteinat a concentration of 100 μg, the group 2 was the positive control andreceived the commercial vaccine. Group 3 was the negative control and,therefore, received PBS. Blood samples were collected on days 0, 32, 81and 145 post-vaccination to obtain the serum.

To determine the humoral immune response of the immunized pigs,commercial 96-well microplates were used. The microplates were coatedwith the solution of the rCap-PCV-2 (1.125 μg/mL) diluted in carbonatebuffer 0.05 M overnight at 4° C. After this period, the plates werewashed with PBS containing Tween 20 (PBS/T—NaCl 137 mM, KCl 2.7 mM,Na₂HPO₄ 10 mM, KH₂PO₄ 2 mM, Tween 20 0.05%; pH 7.2), blocked withblocking solution (PBS/T containing BSA) under stirring of 500 rpm andat 37° C. in incubator for commercial microplate. Then the plates wereincubated with sera samples diluted (1:800) in dilution solution (PBS/Tcontaining BSA 0.5%), with each sample being tested in triplicate. Afterwashing again, the plates were incubated with secondary antibody (swineanti-IgG conjugated with peroxidase produced in rabbit, Sigma), dilutedin dilution solution. Subsequently, the plates were washed again and forthe colorimetric reaction a solution containing the chromogenicsubstrate was added [10 mL of citrate buffer 0.1 M; pH 5;o-Phenylenediamine 4 mg (OPD); 5 μL of hydrogen peroxide (H₂O₂) 30%].The reaction was incubated for 10 minutes and paralyzed with sulfuricacid solution 1.5 M (H₂SO₄). The presence of antibody was determined byreading the absorbance at 492 nm (D0492 nm) in commercial microplatereader. The results were analyzed by the Prisma statistical program andby the Newman-Keuls multiple comparison test.

In the first blood sample (day 0), which was before applying thevaccine, the three groups showed homogeneous results with respect to theantibody levels. In the second collection (32 days post-vaccination),there was no statistically significant difference between the vaccinegroup and control group. In the third (81 days post vaccination) andfourth collections (145 post-vaccination), the results were similar,wherein the vaccine candidate generated a higher antibody responsecompared to the positive and negative control groups (FIG. 9).

Farm II

The trial II was conducted in a commercial farm of complete cyclenaturally infected with PCV2. The study was conducted in the region ofZona da Mata—MG, respecting the management adopted by the farm and inaccordance with the ethical standards of animal testing and requirementsof the Ethics Committee on Animal Use (CEUA) of the UFV. It was used 120swine, males and females, with commercial genetic of approximately 23days old, divided into 4 groups of 30 animals. At 23 days of age, thepiglets were identified with an ear tag proper for swine, and thereafterweighed for the groups division. This division was made by randomexperimental lineation, so that all groups had similar average weight.

The vaccination was performed in a single 1 mL dose intramuscularly atweaning (animals at approximately 3 weeks of age). The group 1 was thenegative control group and the animals were vaccinated with PBS 1 mL(saline solution). The group 2 was considered a positive control groupin which animals received 1 mL of a commercial vaccine. The group 3 wasthe group that received 1 mL of vaccine prepared from the partiallypurified soluble fraction (150 μg of antigen). The group 4 was the groupthat received 1 mL of vaccine test purified with ammonium sulfate (150μg of antigen).

Blood samples were collected through the jugular vein using sterileneedles in blood collection tubes of 8 mL with clot activator, beingstored in the first hours at room temperature to clot retraction andobtaining serum. The sample collections were performed at 0, 42, 93, and135 days after vaccination. Antibody profiles were performed by indirectELISA as described in the experiment of Farm I. The results wereanalyzed by the statistical software Prisma and the Newman-Keulsmultiple comparison test.

The first blood collection was performed at the time of weaning at 23days old (0 days of vaccination), prior to vaccination, and at thattime, the piglets showed results homogenous regarding the antibodylevels. In the second collection, which occurred at 42 dayspost-vaccination with the animals averaging 65 days of age, there was astatistical difference in antibody levels between treatments. Groups 2,3, and 4 were statistically different from group 1. In addition, groups3 and 4 were statistically different from Group 2. In the thirdcollection, 93 days after vaccination, with the animals at an average of116 days of age, Groups 2, 3, and 4 were statistically different fromgroup 1. The groups 3 and 4 differ significantly from group 2. In thefourth and last collecting, at 135 days post vaccination and animals at158 days of age, groups 3 and 4, prepared from the vaccine candidatewere statistically different from group 1, whereas there was nostatistical difference at this stage between the positive and negativecontrols.

1. A recombinant antigen of the porcine circovirus 2 which is the SEQ IDNO:
 02. 2. A recombinant antigen, which comprises the product of theoligomerization of SEQ ID NO: 02 in the form of virus-like particles(VLPs) of the PCV-2.
 3. Vaccine formulations which comprise the antigenas defined in claim 1, associated with pharmaceutically acceptableadjuvants.
 4. Uses of the antigen as defined in claim 1, which isapplied in the production of vaccine compounds.
 5. Use of the vaccineformulations defined in claim 3, which is for the control of diseasesrelated to the PCV-2.
 6. Vaccine formulations which comprise the VLPs asdefined in claim 2 associated with pharmaceutically acceptableadjuvants.
 7. Uses of the antigen as defined in claim 2 which is appliedin the production of vaccine compounds.
 8. Use of the vaccineformulations as defined in claim 6 which is for the control of diseasesrelated to the PCV-2.